Tbx5 is Required for Avian and Mammalian Epicardial Formation and Coronary Vasculogenesis.

Rationale: Holt-Oram syndrome (HOS) is an autosomal dominant heart-hand syndrome caused by mutations in the TBX5 gene. Overexpression of Tbx5 in the chick proepicardial organ (PEO) impaired coronary blood vessel formation. However, the potential activity of Tbx5 in the epicardium itself, and Tbx5\u27s role in mammalian coronary vasculogenesis, remains largely unknown. Objective: To evaluate the consequences of altered Tbx5 gene dosage during PEO and epicardial development in the embryonic chick and mouse. Methods and Results: Retroviral-mediated knockdown or upregulation of Tbx5 expression in the embryonic chick PEO as well as proepicardial-specific deletion of Tbx5 in the embryonic mouse (Tbx5(epi-/-)) impaired normal PEO cell development, inhibited epicardial and coronary blood vessel formation and altered developmental gene expression. The generation of epicardial-derived cells (EPDCs) and their migration into the myocardium was impaired between embryonic day (E) 13.5-15.5 in mutant hearts due to delayed epicardial attachment to the myocardium and subepicardial accumulation of EPDCs. This caused defective coronary vasculogenesis associated with impaired vascular smooth muscle cell recruitment, and reduced invasion of cardiac fibroblasts and endothelial cells into myocardium. In contrast to wildtype hearts that exhibited an elaborate ventricular vascular network, Tbx5(epi-/-) hearts displayed a marked decrease in vascular density that was associated with myocardial hypoxia as exemplified by HIF1α upregulation and increased binding of Hypoxyprobe-1. Tbx5(epi-/-) mice with such myocardial hypoxia exhibited reduced exercise capacity compared to wildtype mice. Conclusions: Our findings support a conserved Tbx5 dose-dependent requirement for both proepicardial and epicardial progenitor cell development in chick and mouse coronary vascular formation

Cardiac muscle cell formation after development of the linear heart tube

After the development of the linear heart tube, additional myocardium is formed leading to the muscular mantle around the caval and pulmonary veins and the muscular septa in the embryonic heart. Here, we report the results of our in vivo and in vitro studies of this late myocardium-generating process in the mouse. By using an immunohistochemical approach, we determined that myocardium formation starts around embryonic day 12 in the dorsal mesocardium. In subsequent stages of development, the process extends downstream into the intracardiac mesenchymal tissues of the atrioventricular canal and outflow tract and upstream into the extracardiac mediastinal mesenchyme embedding the pulmonary and caval veins. Given the spatiotemporal pattern of myocardium formation, we applied a three-dimensional in vitro explant culture assay to investigate the myocardium-generating potential of the different cardiac compartments. We determined that this potential is stage- and mesenchyme-dependent. This latter finding suggests an important role for mesenchyme in myocardium formation after the development of the linear heart tub

Recruitment of intra- and extracardiac cells into the myocardial lineage during mouse development

The tubular heart differentiates from the bilateral cardiac fields in the splanchnic mesoderm. The expression of smooth muscle proteins has been shown to accompany the early phases of cardiac muscle formation. In this study we show that during elongation of the arterial pole of the mouse linear heart tube, alpha-smooth muscle actin (alpha-Sma) expression extends in the area that has been shown to become recruited into the myocardial lineage, but does not yet express myocardial markers. These data suggest that alpha-Sma identifies mesodermal cells that during subsequent development will be recruited into the myocardial lineage. Myocardium formation is not only observed at the arterial pole, but also at the venous pole and in the intracardiac mesenchyme. This results in the formation of the caval and pulmonary myocardium, the smooth-walled atrial myocardium, the myocardial atrioventricular septum, and the myocardial outlet septum. To determine whether recruitment into the myocardial lineage also takes place in these regions, the spatiotemporal pattern of expression of alpha-Sma and of the myocardial markers sarcoplasmatic reticulum calcium ATPase (Serca2a), alpha-myosin heavy chain (Mhc), and beta-Mhc were examined. We show that prior to the expression of myocardial markers, alpha-Sma is expressed in these regions, which suggests that these mesodermal cells become recruited into the cardiac lineage after formation of the linear heart tub

In vivo and in vitro approaches reveal novel insight into the ability of epicardium-derived cells to create their own extracellular environment

\u3cp\u3eHuman epicardium-derived cells (hEPDCs) transplanted in the NOD-SCID mouse heart after myocardial infarction (MI) are known to improve cardiac function, most likely orchestrated by paracrine mechanisms that limit adverse remodeling. It is not yet known, however, if hEPDCs contribute to preservation of cardiac function via the secretion of matrix proteins and/or matrix proteases to reduce scar formation. This study describes the ability of hEPDCs to produce human collagen type I after transplantation into the infarct border zone, thereby creating their own extracellular environment. As the in vivo environment is too complex to investigate the mechanisms involved, we use an in vitro set-up, mimicking biophysical and biochemical cues from the myocardial tissue to unravel hEPDC-induced matrix remodeling. The in vivo contribution of hEPDCs to the cardiac extracellular matrix (ECM) was assessed in a historical dataset of the NOD-SCID murine model of experimentally induced MI and cell transplantation. Analysis showed that within 48 h after transplantation, hEPDCs produce human collagen type I. The build-up of the human collagen microenvironment was reversed within 6 weeks. To understand the hEPDCs response to the pathologic cardiac microenvironment, we studied the influence of cyclic straining and/or transforming growth beta (TGFβ) signaling in vitro. We revealed that 48 h of cyclic straining induced collagen type I production via the TGFβ/ALK5 signaling pathway. The in vitro approach enables further unraveling of the hEPDCs ability to secrete matrix proteins and matrix proteases and the potential to create and remodel the cardiac matrix in response to injury.\u3c/p\u3

BMP and FGF regulate the differentiation of multipotential pericardial mesoderm into the myocardial or epicardial lineage

Proepicardial cells give rise to epicardium, coronary vasculature and cardiac fibroblasts. The proepicardium is derived from the mesodermal lining of the prospective pericardial cavity that simultaneously contributes myocardium to the venous pole of the elongating primitive heart tube. Using proepicardial explant cultures, we show that proepicardial cells have the potential to differentiate into cardiac muscle cells, reflecting the multipotency of this pericardial mesoderm. The differentiation into the myocardial or epicardial lineage is mediated by the cooperative action of BMP and FGF signaling. BMP2 is expressed in the distal IFT myocardium and stimulates cardiomyocyte formation. FGF2 is expressed in the proepicardium and stimulates differentiation into the epicardial lineage. In the base of the proepicardium, coexpression of BMP2 and FGF2 inhibits both myocardial and epicardial differentiation. We conclude that the epicardial/myocardial lineage decisions are mediated by an extrinsic, inductive mechanism, which is determined by the position of the cells in the pericardial mesoder

Regional differences in WT-1 and Tcf21 expression during ventricular development: implications for myocardial compaction

<div><p>Background</p><p>Morphological and functional differences of the right and left ventricle are apparent in the adult human heart. A differential contribution of cardiac fibroblasts and smooth muscle cells (populations of epicardium-derived cells) to each ventricle may account for part of the morphological-functional disparity. Here we studied the relation between epicardial derivatives and the development of compact ventricular myocardium.</p><p>Results</p><p>Wildtype and Wt1^CreERT2/+ reporter mice were used to study WT-1 expressing cells, and Tcf21^lacZ/+ reporter mice and PDGFRα^-/-;Tcf21^LacZ/+ mice to study the formation of the cardiac fibroblast population. After covering the heart, intramyocardial WT-1+ cells were first observed at the inner curvature, the right ventricular postero-lateral wall and left ventricular apical wall. Later, WT-1+ cells were present in the walls of both ventricles, but significantly more pronounced in the left ventricle. Tcf21-^LacZ + cells followed the same distribution pattern as WT-1+ cells but at later stages, indicating a timing difference between these cell populations. Within the right ventricle, WT-1+ and Tcf21-lacZ+ cell distribution was more pronounced in the posterior inlet part. A gradual increase in myocardial wall thickness was observed early in the left ventricle and at later stages in the right ventricle. PDGFRα^-/-;Tcf21^LacZ/+ mice showed deficient epicardium, diminished number of Tcf21-^LacZ + cells and reduced ventricular compaction.</p><p>Conclusions</p><p>During normal heart development, spatio-temporal differences in contribution of WT-1 and Tcf21-^LacZ + cells to right versus left ventricular myocardium occur parallel to myocardial thickening. These findings may relate to lateralized differences in ventricular (patho)morphology in humans.</p></div

Ventricular compaction and wall thickness in sequential stages E11.5–15.5.

<p>A-E. Myocardial staining for myosin light chain 2a (MLC-2a) shows myocardial compaction and thickening of the anterior part of right ventricle (RV). F-J: Myocardial compaction and thickening of posterior part of RV. K-O: Myocardial compaction and thickening of the left ventricle (LV). The LV wall is thicker than RV, and the RV posterior wall appears thicker than anterior (compare sections E, J and O). P-R depict the myocardial wall thickness measurements of the RV and LV. Two distinction can be made in the measurements. One is the <i>anatomical region</i> of each ventricle: the anterior portion of the RV, the posterior portion of the RV or the posterior portion of the LV. The second distinction is the <i>location in the myocardial wall</i>: the lateral wall or the apical wall. P. The RV shows a significantly thicker posterior than anterior wall at E15.5. Q,R: RV and LV wall thickness. From E13.5, LV lateral wall (Q) is significantly thicker than RV, except for the latest stage (E15.5). For the apical wall (R) this difference is significant from E12.5. Grey line with circle (°) represents significant difference in LV thickness between E11.5-E13.5 and E14.5-E15.5, showing a gradual growth during development. Grey line with square (□) represents significant difference in RV thickness between 11.5–14.5 and E15.5, indicating a gradual ventricular growth during development. Grey line with alpha (α) represents significant difference in LV thickness between E11.5-E12.5 and E15.5. *p<0.05; **p<0.01; ***p<0.001. Bars: 50 μm.</p

Regional differences in intramyocardial WT-1 expression at E13.5.

<p>A-D: Control, transverse sections. A. Anterior section, stained for WT-1. B: Enlargement of the box in A with few WT-1+ cells in the right ventricle (RV) and more WT-1+ cells in the left ventricle (LV). C. Posterior section, stained for WT-1. D: Enlargement of RV in D with more WT-1+ cells than anterior and enlargement of the LV with overall more WT-1+ cells than in the RV (compare with panel B). E-J: fluorescence sequential sagittal sections from RV (E-F, H-I) towards LV (G,J) double stained for WT-1 (green) and cTnI (red). Arrows indicate areas with abundant WT-1+ cells. Anterior (A) and posterior (P) are indicated. K-N: Tcf21^lacZ/+ mouse sections stained for LacZ in blue. K. Anterior section of the heart. L: Enlargement of RV with no LacZ+ cells and of LV with sporadic WT-1+ cells (arrowheads). M. Posterior section of the heart. N: Enlargement RV with sporadic LacZ+ cells (arrowheads) and of LV with overall more LacZ+ cells than in the RV (arrowheads). Bars: A,C,E,G: 200 μm, B,D,F,H: 50 μm, I-N: 100 μm.</p

WT1-derived cells and Tcf21^lacZ/+ show regional differences in intramyocardial distribution of at E14.5.

<p>A-E: WT1^CreERT2/+; Rosa26^fsLz/+ mouse section (A,B,D) and control WT-1 staining (C,E) showing differences between RV and LV in WT-1+ cells. Red tissue in A,B,D indicates β-gal staining. A is an overview of RV and LV, B and D are enlargements of RV (B) and LV (D). C and E show WT-1+ cells comparable to B and D. F-I: H: Tcf21^lacZ/+ mouse section stained for LacZ in blue. Anterior (Ant) and posterior (Pos) are indicated. The upper box at the RV (H) corresponds to the enlargement in F of the anterior RV, the lower box at the RV (H) corresponds to the enlargement (G) of the posterior RV. The box of the LV in H corresponds to enlargement (I). Tcf21^lacZ/+ demonstrates the same distribution pattern, i.e. more lacZ+ cells in LV than RV (compare G with I) and more lacZ+ cells in posterior part of RV than anterior (compare G with F). Bars: I,N: 200 μm, other bars: 50 μm.</p